The present invention relates to the drug screening, diagnostic, and synthesis uses of the first recombinant derived adult human liver flavin-containing monooxygenase (form 3), also referred to as adult human liver FMO (form 3) or HLFMO 3.
The mammalian flavin-containing monooxygenase (FMO, EC 1.14.13.8, Dimethylaniline N-oxidase) is a widely distributed enzyme that catalyzes the NADPH-dependent oxygenation of a wide variety of nucleophilic nitrogen-, sulfur-, and phosphorous-containing drugs, chemicals, and xenobiotics (Cashman, Chem. Res. Toxicol. 8:165-181 (1995); Ziegler, Enzymatic Basis of Detoxication 1:201-225 (1980); and Ziegler, Drug Metab. Rev. 6:1-32 (1988)). To dates, many of the investigations examining hepatic FMO have been performed with animal tissues, possibly because of the thermal instability of adult human liver FMO preparations. In contrast to adult human liver cytochromes P-450, almost nothing is known about the structure of adult human liver FMO. Adult human liver FMO has been designated FMO3 (Lawton et al., Arch. Biochem. Biophys. 308: 254-257 (1994)). A few studies with adult human liver microsomes have demonstrated FMO-like enzyme activity (Gold et al., Xenobiotica 3:179-189 (1973) Lemoine et al., Arch. Biochem. Biophys. 276:336-342 (1990); McManus et al., Drug Metab. Dispos. 15:256-261 (1987)) and immunoreactivity with the antibody against pig liver FMO1 (Lemoine et al., Arch. Biochem. Biophys. 276:336-342 (1990); Dannan et al., Mol. Pharmacol. 22:787-794 (1982)). Dimethylaniline N-oxygenation was observed in adult (Gold et al., Xenobiotica 3:179-189 (1973)) and fetal (Rane, Clin. Pharmacol. Ther. 15:32-38 (1973)) human liver microsome preparations. In contrast to dimethylaniline N-oxygenation, which was observed in both kidney and liver tissues, imipramine N-oxygenation was only observed in microsome preparations from human kidney, but not from human liver (Lemoine et al., Arch. Biochem. Biophys. 276:336-342 (1990)). The conclusion from these studies was that FMO was present in human liver tissue, albeit with low specific activity and possibly as multiple enzyme forms. This has been verified with the cloning of five forms of FMO cDNA from human liver cDNA libraries (Phillips et al., Chem. Biol. Interact. 96:17-32 (1995)).
In animals, FMO has been reported to be present as at least one pulmonary form (Williams et al., Biochem. Biophys. Res. Commun. 124:116-122 (1984); Tynes et al., Biochem. Biophys. Res. Commun. 126:1069-1075 (1985)) and as two or more hepatic forms (e.g., forms 1 and 3) (Yamada et al., Arch. Biochem. Biophys. 280:305-312 (1990); Ozols, J. Biol. Chem. 265:10289-10299 (1990)). It is more recently recognized that FMOs are present in multiple tissues and xe2x80x9chepaticxe2x80x9d and xe2x80x9cpulmonaryxe2x80x9d forms are misnomers. In rabbit liver, form 1 and 3 are only 55% identical to one another, but the amino acid sequence identity between hog liver FMO1 and rabbit liver FMO form 1 is approximately 87% (Ozols, Arch. Biochem. Biophys. 290:103-115 (1991)). Although studies are limited, forms 1 and 3 FMO apparently differ in many important properties including substrate specificity (Yamada et al., Arch. Biochem. Biophys. 280:305-312 (1990)), enzyme stability (Ozols, Arch. Biochem. Biophys. 290:103-115 (1991)) and other physical properties.
For example, hepatic form 1 FMO activity is stimulated by primary aliphatic alkylamines and form 1 FMO catalyzes the N-oxygenation of secondary and tertiary amines (Ziegler, Enzymatic Basis of Detoxication 1, 201-225 (1980)). In contrast, form 3 FMO apparently N-oxygenates primary aliphatic alkylamines as well as secondary and tertiary amines (Yamada et al., Arch. Biochem. Biophys. 280:305-312. (1990)). Aliphatic primary amines are sequentially N-oxygenated by FMO3 to hydroxylamine and oximes. The pharmacological activity of these metabolities are largely unknown but if FMO3 catalyzes efficient oxime formation from endogenous amines, this could be important in cellular homeostasis. Abnormal amine metabolism by FMO3 could be important in numerous disease states that are associated with abnormal amine metabolism. Some aliphatic tertiary amines such as chlorpromazine are preferentially N-oxygenated by form 3 FMO (Yamada et al., Arch. Biochem. Biophys. 280:305-312 (1990)) but a detailed description of animal FMO3 activity has not been described.
FMO has been purified to homogeneity from a number of sources (Ziegler, Drug Metab. Rev. 19:1-32 (1988)) and it is the pig liver enzyme (FMO form I) which has been the subject of the most extensive studies. Using probes directed against the pig liver FMO and using a fetal human liver cDNA library, a cDNA encoding a FMO has been cloned (Dolphin et al., J. Biol. Chem. 266:12379-12385 (1991)). Thus, fetal human liver flavin-containing monooxygenase (FMO) shares approximately 86% identity with pig liver FMO and 87% identity with rabbit liver FMO form I deduced from the cDNA data (ibid.). Fetal human liver FMO has been designated form 1. Substrate specificity differences are apparent for hepatic form 1 and 3 FMOs from in vitro animal liver enzyme studies (Yamada et al., Arch. Biochem. Biophys. 280:305-312 (1990)), but almost nothing is known about the human liver enzymes.
A number of studies have shown that adult human liver microsomes are capable of tertiary amine N-oxygenation (Gold and Ziegler, Xenobiotica 3:179-189 (1973); McManus et al., Drug Metab. Dispos. 15:256-261 (1987); Lemoine et al., Arch. Biochem. Biophys. 276:336-342 (1990); Rane, Clin. Pharmacol. Ther. 15:32-38 (1973)) and thiobenzamide S-oxygenation (McManus et al., Drug Metab. Dispos. 15:256-261 (1987)).
Adult human liver FMO-dependent N- and S-oxygenation activity is quite thermally labile and activity is maximal at pH 8.4 (Gold and Ziegler, supra; McManus et al., supra; and Lemoine et al., supra, although considerable intersample variation has been observed. Most physical properties of animal FMOs are shared by human liver FMO forms although differences in substrate specificity have not been extensively examined. For example, human liver microsomes did not N-oxygenate imipramine even though imipramine was an excellent substrate for pig liver FMO form I (Lemoine et al., supra). Immunoquantitation of human liver FMO has relied on antibodies directed against animal FMOs. Thus, polyclonal antibodies prepared against pig liver FMO recognized a 60,000 Da human liver protein, although the immunoblot was characterized as very faint. Antisera raised against rat liver FMO recognized an adult human kidney protein, but did not recognize anything in the adult human liver (Lemoine et al., supra (1990)). This is another indication that multiple forms of FMO are present in the adult human liver and kidney.
For over 25 years, the literature has described a few people who, instead of N-oxygenating trimethylamine (TMA) to the polar, readily excreted trimethylamine N-oxide (TMANO), excreted large amounts of unmetabolized TMA in the urine and secreted the volatile and malodorous TMA in their breath, sweat and skin (Humbert, et al., Lancet i:770-771 (1970); Higgins et al., Biochem. Med. 6:392-396 (1972); Danks et al., N. Engl. J. Med. 25:962 (1976)). TMA smells like the essence of rotting fish and people who suffer from this apparent metabolic disorder have what is referred to as the xe2x80x9cfish-odor syndrome.xe2x80x9d In humans, trimethylaminuria is an autosomal recessive disorder involving deficient N-oxygenation of TMA (Al-Waiz et al., Br. J. Clin. Pharmacol. 25:664p-665p (1993); Ayesh, et al., Br. Med. J., 655-657 (1993); Ayesh and Smith, Pharmacol. Ther. 45:387-401 (1990)). Normally, over 95% of a dose of TMA from dietary sources or otherwise is converted to TMANO that is excreted in the urine. The ability to N-oxygenate TMA is apparently distributed polymorphically (at least in some Caucasian populations evaluated thus far) and people with xe2x80x9cfish-odor syndromexe2x80x9d are apparently homozygous for an allele that determines an individuals ability to carry out the N-oxygenation reaction (Ayesh et al., Br. J. Clin. Pharmacol. 25:664p-665p (1993). The molecular defect in trimethylaminuria has not yet been defined although it has been attributed to a deficiency in human FMO1 (Dolphin et al., J. Biol. Chem. 266:12379-12385 (1991); Dolphin et al., Biochem J. 287:261-267 (1992). It is now known, however, in contrast to what has been previously described (Dannan and Guengerich, Mol. Pharmacol. 22:787-794 (1982)), that human FMO1 is not expressed to a measurable extent in adult human liver and it is adult humans that have been associated with the disease. It is not likely that other monooxygenases form TMANO from TMA, based on existing studies (Gut and Conney, Drug Metab. Drug Interacts. 9:201-208 (1991) and the fact that TMA is a very good substrate for FMO from rat liver (Horori and Benoit, Biochem. Biophys. Res. Commun. 212:820-826 (1995). As described herein, cDNA-expressed human FMO3 is a good catalyst for the formation of TMANO from TMA in vitro. It is likely that human FMO3 is largely responsible for the N-oxygenation of TMA.
The deficiency of human FMO3 as an explanation for trimethylaminuria is probably more prevalent than previously realized (Treacy et al., J. Inher. Dis. 18:306-312 (1995) and the fish-odor syndrome is a major social handicap to patients who are usually anxious to obtain treatment. In addition to the psychosocial consequences of trimethylaminuria (i.e., anxiety, clinical depression, paranoia, suicidal personality and addiction to cigarettes, alcohol and drugs) on drug and endogenous amine metabolism as a result of altered or deficient human FMO3 activity (Chen and Aiello Am. J. Med. Genet. 45:335-339 (1993)), the possibly more important consequences of trimethylaminuria is that it may foretell about other more serious human diseases. For example if human FMO3 is involved in biogenic or other endogenous amine metabolism, a deficiency of human FMO3 may have profound consequences for a wide spectrum of diseases related to abnormal amine metabolism including cardiovascular disease and associated disorders, hypertension, and central nervous system diseases including but not limited to depression, stress, epilepsy, Huntington""s, Parkinson""s and Alzheimers disease and infectious diseases.
In adult human liver microsomes, in addition to FMO there are numerous other enzymes present including esterases and other monooxygenases. In some cases the other esterases and monooxygenases compete with the adult human liver FMO for substrate activity. For example, an esterase that converts a methyl ester to a carboxylic acid could make that compound unusable as a substrate for the FMO. Other monooxygenases present in the adult human liver could also compete with FMO for substrate activity. Microsomes also generate hydrogen peroxide and alkyl peroxide. Peroxide generated by microsomes could oxidize substrates of FMO such as sulfur, nitrogen, and phosphorous-containing chemicals. Therefore, the presence of such peroxides and other monooxygenases make it difficult to determine the true enzymatic activity and substrate specificity for adult human liver FMO. For this FMO to have any practical use in the research and industrial areas, it must be obtained in a form that is free of human liver microsomal monooxygenases and peroxides generated from these preparations.
The present invention provides adult human liver flavin-containing monooxygenase (form 3) in substantially pure form. The invention also includes mutants, variants, and fusion products of adult human liver flavin-containing monooxygenase (form 3). The invention also concerns a DNA sequence, and fragments and derivatives thereof, encoding adult human liver flavin-containing monooxygenase (form 3), and host cells involved in the expression of the adult human liver flavin-containing monooxygenase (form 3).
Another aspect of the invention includes methods for in vitro screening of compounds for biological or pharmacological activity. These methods include incubating a monooxygenase with the compound to detect the amount of oxygen consumed, the amount of NADPH consumed, or products formed.
A further aspect of the invention involves methods for detecting cancer in liver cells. This involves generating antibodies that react with the fetal human liver flavin-containing monooxygenase (form 1) and not FMO (form 3). Another method for detecting cancer in liver cells is the use of oligonucleotide probes that bind to the mRNA of the fetal human liver flavin-containing monooxygenase (form 1) and not FMO (form 3) gene. An additional method for detecting liver cancer uses the technique of PCR to amplify the cDNA of the fetal human liver flavin-containing monooxygenase (form 1) gene for hybridization with a probe.
Another aspect of the invention includes a method of selectively oxidizing a nucleophilic compound by incubating a monooxygenase with the nucleophilic compound.
An additional aspect of the invention involves a method of producing a nucleophilic compound with a center of chirality by incubating a monooxygenase with a substrate. This can be coupled in an asymmetric chemi-enzymatic synthesis of a chiral chemical or drug by reacting the resulting oxidized substrate with a strong base and an electiophilic compound.
Another aspect of the invention includes a method of assembling a native or active protein or peptide by incubating a monooxygenase capable of forming disulfides with an unfolded protein or peptide.
A further aspect of the invention involves a method of expressing a monooxygenase in a cDNA expression system to act as a catalyst for the renaturation of proteins or peptides by facilitating disulfide bond formation and protein folding.